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International Health Institute and Departments of Pediatrics, Molecular Microbiology and Immunology
Pathology and Laboratory Medicine, Brown University, Providence, Rhode Island
Walter Reed Army Institute of Research, Silver Spring, Maryland
Seattle Biomedical Research Institute, Seattle, Washington
Previously, we collected plasma from 143 male volunteers residing in an area of western Kenya where Plasmodium falciparum is holoendemic. Volunteers were cured of current malaria infection by use of drugs, blood was collected 2 weeks after treatment, and blood films were collected weekly for 18 weeks. We identified and pooled plasma from the 10 most resistant individuals (RP) and the 7 most susceptible individuals (SP) and used these pools in a differential screen of a P. falciparum cDNA expression library. We screened 550,000 clones and identified 7 clones that were uniquely recognized by RP but not by SP. Two clones encoded a C-terminal region polypeptide from rhoptry-associated membrane antigen (RAMA-pr), a recently described RAMA. We measured antiRAMA-pr antibody levels in plasma obtained 2 weeks after treatment. Individuals with detectable immunoglobulin G1 antiRAMA-pr (n = 24) had fewer positive blood films (odds ratio, 1.7 [95% confidence interval, 1.212.44]; P < .003), 43% lower density of parasitemia (P < .02), and prolonged time to reinfection (P < .0027), compared with individuals without detectable antibody levels (n = 115), after known determinants of resistance were accounted for. In summary, RAMA-pr is a rationally identified vaccine candidate that is preferentially recognized by antibodies produced by humans with a high level of naturally acquired resistance to P. falciparum infection.
The enormous global burden of malaria has increased in recent years, and the international health community has prioritized the development of a vaccine for the deadly human malaria parasite Plasmodium falciparum [1]. Discovery of antigens for malaria vaccines has traditionally relied on vaccination experiments in animal models [24], the identification of antigens with specific features (such as localization to the surface of the parasite) [5], or expression during specific stages of the parasite life cycle [6]. Subsequent to discovery of antigens, cross-sectional and longitudinal analyses of human immune responses to these candidates have been used to evaluate their role as targets of protective human immunity [7, 8]. However, naturally acquired immune responses have generally not been exploited as tools at the stage of discovery of antigens for vaccines.
Residents of areas where P. falciparum is endemic develop protective immunity that limits parasitemia and disease [9, 10]. Expression of naturally acquired immunity is highly variable [7, 11], is species specific [12, 13], and requires both repeated exposure [14] and host pubertal development for maximal expression [11, 15]. Evidence from passive-transfer experiments in humans indicates that antibodies against blood-stage parasites can block erythrocyte invasion, increase splenic clearance, and mediate, in part, naturally acquired resistance in humans [1618]. Therefore, naturally acquired human resistance to malaria represents an attractive and underused model for identification of new vaccine candidates.
We hypothesize that variability in antibody recognition of unique parasite proteins mediates, in part, the heterogeneity observed in the intensity and frequency of reinfection with P. falciparum after treatment. Using plasma and parasitologic data from an immunoepidemiologic reinfection study, we differentially screened a P. falciparum cDNA expression library using plasma pooled from resistant versus susceptible individuals. We identified several parasite proteins that were uniquely recognized by resistant individuals and present the characterization of one of these clones here.
SUBJECTS, MATERIALS, AND METHODS
Study population.
This study is a secondary analysis conducted with data and blood samples that were collected in 1997 as part of a treatment-reinfection study [7, 11]. Volunteers were residents of subsistence farming, falciparum-endemic villages in western Kenya, north of Lake Victoria. The entomological inoculation rate in this area can exceed 300 infectious bites/year [19]. The present study was approved by the institutional review boards of the Walter Reed Army Institute of Research, the Kenyan Medical Research Institute, and Brown University.
After providing informed consent, 144 males aged 1235 years were entered into the study at the beginning of the high transmission season (April) in 1997. Detectable parasitemia was eradicated in 143 of the 144 participants by use of quinine sulfate (10 mg/kg twice daily for 3 days) and doxycycline (100 mg twice daily for 7 days). One volunteer remained parasitemic during the week after treatment and was removed from the analysis. Immunologic and epidemiologic analyses of this cohort have been reported elsewhere [7, 11, 20, 21].
Malaria assessment.
Thick and thin blood films were obtained from each volunteer before treatment and then weekly for 18 weeks after treatment, to quantify reinfection. Each film was interpreted by 2 microscopists, and the mean of the 2 values was recorded.
Trained field workers visited volunteers each day to ascertain their well-being and to minimize unreported use of antimalarials. Sick volunteers were transported to the study clinic, and those with positive blood films and clinical symptoms suggestive of acute malaria were treated with 3 tablets of Fansidar (Hoffman-La Roche). Data on blood films from individuals receiving Fansidar were censored at the time of treatment, as described elsewhere [7]. Analysis of censored and uncensored data produced similar results; therefore, we present results for censored data only.
Entomology measurements.
The intradomicillary female anopheline abundance was measured weekly for 18 weeks in each volunteer's domicile by use of the Daytime Resting Indoors (DRI) method [22], as described elsewhere [7].
Blood collection.
Two weeks after treatment with quinine and doxycycline, volunteers donated 10 mL of blood into heparinized tubes. Within 4 h of collection, samples were centrifuged, and plasma was aliquoted and stored at -70°C for subsequent analysis.
Clinical assays.
Hemoglobin electrophoresis and dehydroepiandrosterone sulfate (DHEAS) assays were performed as described elsewhere [11].
Selection of resistant and susceptible individuals.
Using the mean parasite density of the 18 posttreatment blood films, we identified and pooled plasma from the 10 most resistant individuals (RP) and the 7 most susceptible individuals (SP), after matching for the potential confounding variables of age, mosquito exposure, and hemoglobin phenotype (AA vs. AS-sickle trait). Matching was not performed for DHEAS levels, because this hormone may lie in the causal pathway of protective antibody production [11].
Differential screening of cDNA library.
We obtained a P. falciparum merozoite-stage cDNA expression library (MRA-299) from MR4. We prepared and screened duplicate filters from this library with RP and SP, in accordance with the manufacturer's directions (Stratagene), using a polyvalent antihuman immunoglobulin (Sigma) for detection. Positive clones were confirmed by back-mixing experiments in which the putative positive clone was mixed with negative plaques at a known proportion, plated, and probed with RP and SP. True-positive clones reacted in the expected proportion with RP but not with SP.
Expression of recombinant proteins.
Clones uniquely reactive with RP were excised with helper phage and sequenced, and the open-reading frame (ORF) was amplified by polymerase chain reaction (PCR) and cloned in a ligation-independent fashion into the high-expression plasmid pET32/Xa (Novagen), as described elsewhere [23]. This expression system produces a fusion protein with an N-terminus thioredoxin tag that enhances solubility of the expressed antigens and a C-terminus hexa-histidine tag to assist in purification. The clones were transformed into the expression hosts BL21(DE3) and BLR(DE3) (Novagen). The transformants were grown in Luria broth supplemented with 100 g/mL carbenicillin and 0.05% glucose, at 37°C with shaking at 400 rpm. At mid-log phase (OD600 of 0.5), the temperature was reduced to 20°C, isopropyl--D-thiogalactopyranoside was added (to a final concentration of 1 mmol/L), and the culture was induced for 12 h. After induction, cultures were harvested by centrifugation; resuspended in 10 mmol/L potassium phosphate, 150 mmol/L NaCl, and 10 mmol/L imidazole (pH 8.0); and lysed by sonication.
Recombinant protein purification.
Lysed cells were centrifuged, and the supernatant was filter sterilized. Protein purification was achieved by a 3-step process on fast-performance liquid chromatography equipment (Pharmacia). Briefly, supernatant derived from 3 L of Escherichia coli culture was applied to a Waters AP-1 column containing 7 mL of Ni-NTA Superflow Resin (Novagen). The protein of interest was eluted with a stepped gradient containing increasing concentrations of imidazole. Fractions containing the protein of interest were pooled, buffer exchanged into 10 mmol/L Tris and 1 mmol/L EDTA (pH 8.0), adjusted to 1 mol/L ammonium sulfate, and further purified, by hydrophobic-interaction chromatography on a High Prep 16/10 Phenyl FF column (Pharmacia). Recombinant proteins were eluted with a linear gradient of elution buffer (10 mmol/L Tris and 1 mmol/L EDTA [pH 8.0]). Final purification was achieved by size-exclusion chromatography on a Superdex 200 Prep grade 35/600 BioPilot column (Pharmacia) for a C-terminal region polypeptide from rhoptry-associated membrane antigen (RAMA-pr) and merozoite surface protein (MSP)3 and by anion-exchange chromatography on a Q10 column (BioRad) for MSP-7. Proteins were depleted of endotoxin by use of End-X affinity resin (Cape Cod Associates) overnight at 4°C. Endotoxin depletion was repeated until the lipopolysaccharide concentration in the sample was <100 endotoxin units/mg of protein, as determined by use of a colorimetric limulus amebocyte lysate assay (BioWhittaker). Purification was evaluated on 10% SDS-PAGE gels stained with Gel-Code Blue (Pierce).
Schizont antigen.
Crude schizont antigen was obtained from continuous cultures of a 3D7 strain of P. falciparum. Schizont enrichment was achieved by Plasmagel (Laboratoire Roger Bellon) sedimentation. Enriched fractions were washed 3 times in serum-free parasite culture medium. Fractions were diluted in RPMI 1640, sonicated on ice, and stored at -80°C. The protein concentration was determined by use of a standard bicinchoninic acid protein assay (Pierce).
Generation of RAMA-pr polyclonal antibody.
Before immunization of rabbits, the thioredoxin tag was cleaved from purified RAMA-pr by digestion with Factor Xa. Native N-terminal RAMA-pr was purified from reaction products by anion-exchange chromatography, and its sequence was confirmed by N-terminal sequencing. Rabbits were immunized with 500 g of antigen in complete Freund's adjuvant at baseline and 500 g of antigen in incomplete Freund's adjuvant at 2, 4, and 6 weeks.
Western blot.
Proteins from schizont-infected or uninfected red blood cells (RBCs) were separated by use of 10% SDS-PAGE gels and transferred to nitrocellulose membranes (BioRad). Membranes were blocked in 1% milk PBS (pH 7.4) and 0.5% Tween 20 for 1 h. Membranes were probed with polyclonal antiRAMA-pr or preimmune rabbit sera, detected by use of a polyvalent anti-rabbit antibody conjugated to alkaline phosphatase, and developed with 5-bromo-4-chloro-3-indolyl phospahte/nitro blue tetrazolium (Sigma).
ELISA.
An ELISA was performed to quantify the antibody response to RAMA-pr and total schizont antigen. A total of 50 L of each antigen at 2 g/mL was coated in duplicate on 96-well microtiter plates. A 1 : 1000 dilution of RP, SP, or plasma from a malaria-naive individual was used as the primary antibody. Polyvalent anti-human heavy-chain goat antibody conjugated to alkaline phosphatase (1 : 5000) was used as the secondary antibody. Plates were developed with p-nitro-phenylphosphate (BioRad) and read at 405 nm.
Total and isotype-specific, antigen-specific immunoglobulin titer.
Ninety-six-well Immulon 2HB plates were coated with 50 L of PBS (pH 7.4) containing 2 g/mL recombinant protein overnight at 4°C. Plates were washed 4 times with PBS with 0.05% Tween 20 (PBST) and blocked with 1% milk PBST (MPBST) for 4 h at 37°C. Plasma samples were thawed, spun for 2 min at 16,000 g, diluted in MPBST, transferred to the corresponding well on the antigen-coated plates, and incubated overnight at 4°C. On the basis of initial titration experiments, plasma samples were diluted 1 : 40 for analysis of IgG1, 1 : 80 for analysis of IgG2, 1 : 640 for analysis of IgG3, 1 : 80 for analysis of IgG4, 1 : 1280 for analysis of IgGT, and 1 : 640 for analysis of IgM. Plates were washed 4 times with PBST and incubated for 4 h at 37°C with 100 L of mouse anti-human isotype-specific secondary antibody (Pharmingen) conjugated to biotin at the following dilutions in MPBST: IgG1, 1 : 1000; IgG2, 1 : 1000; IgG3, 1 : 5000; IgG4, 1 : 1000; IgGT, 1 : 25,000; and IgM, 1 : 25,000. Plates were washed 4 times with PBST, and streptavidin conjugated to alkaline phosphatase (100 L in PBST at a 1 : 1000 dilution) was added to each well and incubated for 1 h at 37°C. Plates were developed with p-nitro-phenylphosphate and read at 405 nm. An individual's isotype-specific antibody level was classified as detectable if the mean optical density of the recombinant malarial antigencoated wells exceeded the mean optical density of the recombinant thioredoxincoated wells.
Statistical analysis.
Final analyses were performed by use of SAS (version 8.02; SAS Institute). P < .05 was considered to be statistically significant. Multivariate models were used to examine the relationship between isotype-specific antibody responses to RAMA-pr and resistance to parasitemia, after adjustment for potential confounding variables. For each volunteer, 18 posttreatment blood films were available for analysis. Therefore, repeated-measures models were constructed to account for the lack of independence of multiple within-individual observations. For continuous outcomes, random-intercept models were constructed using Proc Mixed, with volunteer as random effect and an unspecified covariance matrix. For dichotomous outcomes, generalized estimating equation models were constructed using Proc Genmod with a binomial link function, with volunteer as a random effect, an unspecified covariance matrix, and empirically based SEs. Least-square mean values, which represent the mean adjusted for confounding variables, are presented. Time to reappearance of parasitemia was examined with Cox proportional hazards models, with group differences evaluated by use of the log-rank test.
RESULTS
Identification of resistant and susceptible individuals.
Using reinfection data from previous studies [7, 11, 20, 21], we identified and pooled plasma from the 10 most resistant individuals and the 7 most susceptible individuals, after matching for the potential confounding variables of age, mosquito exposure, and hemoglobin phenotype (AA vs. AS-sickle trait). The descriptive data for the resistant and susceptible groups are presented in table 1.
Recognition of unique malarial proteins by resistant plasma.
Pooled resistant and susceptible plasma were used in a differential screen of a P. falciparumasynchronous 3D7 cDNA expression library. We screened 550,000 clones and identified 200 clones that were recognized by both resistant and susceptible plasma, 15 clones that were recognized only by SP, and 7 clones that were recognized only by RP. Clones recognized only by RP were sequenced, and their identity was determined by nucleotide-based BLAST searches of GenBank sequences or EST sequences in the P. falciparum database at http://www.plasmodb.org.
The 7 clones uniquely recognized by antibodies in the RP group encoded MSP-3 (2 clones), P. falciparum erythrocyte membrane protein (PfEMP)2 (2 clones), MSP-7 (1 clone), and a novel clone that we designated "pREM" (2 clones) [24]. Recently, RAMA, a P. falciparum rhoptry protein with identity to pREM, has been described [25]. We have therefore redesignated the RAMA fragment pREM as "RAMA-pr." The duplicate clones encoding MSP-3, PfEMP-2, and RAMA-pr contained partially overlapping cDNA sequences. cDNA sequences from duplicate clones were aligned, and ORFs were identified: MSP-3, 99265 aa [26]; PfEMP-2, 612707 aa [27]; RAMA, 582767 aa; and MSP-7, 117248 aa [25]. These partial ORFs for MSP-3, MSP-7, and RAMA-pr were subcloned into the high-level expression vector pET 32Xa.
Protein expression and purification.
Protein expression was optimized in the permissive E. coli cell lines BL21(DE3) for RAMA-pr and MSP-7 and BLR(DE3) for MSP-3. Recombinant proteins were purified by a 3-step chromatographic process incorporating nickel-chelate, hydrophobic-interaction, and size-exclusion or anion-exchange chromatography. Each recombinant protein was purified to >98% purity, as assessed by SDS-PAGE (figure 1).
Preferential recognition of RAMA-pr, MSP-3, and MSP-7 by resistant plasma.
To quantify antibody responses to the recombinant malarial proteins identified in the screen of the cDNA expression library, we probed antigen preparations made from P. falciparuminfected and uninfected RBCs, recombinant MSP-3, MSP-7, RAMA-pr, and the recombinant fusion partner, thioredoxin, with RP, SP, and plasma from a malaria-naive North American control in an ELISA format (figure 2). Plasma from the malaria-naive control did not recognize thioredoxin or any malarial protein. SP and RP had similar responses to uninfected and infected RBCs and did not recognize thioredoxin. The binding of RP was 3.4-fold greater to MSP-3, 2.5-fold greater to MSP-7, and 1.8-fold greater to RAMA-pr, compared with that of SP.
Translation and expression of RAMA-pr by infected RBCs.
To demonstrate that RAMA-pr was synthesized by erythrocyte-stage parasites, we probed P. falciparuminfected and uninfected RBCs with antiRAMA-pr polyclonal rabbit antisera. AntiRAMA-pr sera, but not preimmune sera, recognized a 50-kDa antigen in infected RBCs but not in uninfected RBCs (figure 3).
Prediction of resistance to P. falciparum reinfection by antiRAMA-pr IgG1 responses.
Isotype-specific immunoglobulin responses to recombinant antigens were assessed in each member of the cohort. IgGT responses to MSP-3, MSP-7, and RAMA-pr were detectable in 24.5%26.6% of individuals (table 2). IgG3 responses to these antigens were detected in the greatest proportion of individuals (55.2%66.4%), whereas IgG1 responses were detected in the smallest proportion of individuals (12.6%19.6%).
In multivariate analyses, individuals with detectable antiRAMA-pr IgG1 (n = 24) measured after treatment but before reinfection had a significantly decreased risk of having a positive blood film during the 18-week follow-up period (odds ratio, 1.7 [95% confidence interval, 1.212.44]; P < .003), compared with individuals without detectable antiRAMA-pr IgG1 (n = 115), even after known determinants of resistanceincluding age, DHEAS level, and exposurewere accounted for. In addition, the parasite density of the 18 weekly posttreatment blood films was 43% lower in individuals with detectable antiRAMA-pr IgG1 than in individuals without detectable antiRAMA-pr IgG1, even after age, DHEAS level, and exposure were accounted for (mean ± SE, 1.16 ± 0.1 vs. 0.66 ± 0.2 parasites/200 white blood cells ; P < .02) (figure 4A). Furthermore, individuals with detectable antiRAMA-pr IgG1 had significantly longer times to reinfection during the 18-week follow-up period than did individuals without detectable antiRAMA-pr IgG1, even after age, DHEAS level, and exposure were accounted for (P = .01) (figure 4B).
Individuals with detectable IgG2 antibody to MSP-7 experienced a 33% reduction in parasite density (mean ± SE, 1.16 ± 0.1 vs. 0.78 ± 0.1 parasites/200 WBCs; P = .04). This reduction remained after exposure was accounted for but was not dissociable from the effect of age (data not shown). Isotype-specific antibody responses to MSP-3 were not associated with resistance to reinfection in our population (data not shown).
DISCUSSION
The majority of current falciparum malaria vaccine candidates have been identified on the basis of their surface localization or stage-specific expression or the ability of homologues to confer resistance in animal models. Immunoepidemiologic evaluation in observational human studies has been employed to assess the contribution of specific immune responses to these candidates to naturally acquired resistance to infection.
As an alternative to these approaches, we have employed a vaccine identification strategy based on the differential screening of a parasite cDNA expression library with pooled plasma from humans with high or low levels of naturally acquired resistance to reinfection with P. falciparum. Using this approach, we identified 4 malarial antigensMSP-3, MSP-7, PfEMP-2, and RAMA-prthat were uniquely recognized by antibodies in the plasma of resistant individuals but not of susceptible individuals. Immune responses to several of these candidates have been implicated in mediating resistance in previous work, thus validating our vaccine identification approach.
Immunization with MSP-3 confers protection from P. falciparum challenge in monkeys [28, 29], and cytophilic antibodies against MSP-3 contribute to naturally acquired protection in humans through a monocyte-dependent mechanism [30, 31]. MSP-7, which is a component of the shed MSP-1 complex, does not confer protection after vaccination in a P. yoelii model [32]; however, P. berghei parasites with a deletion of the MSP-7 gene demonstrate attenuated growth in vitro [33]. The association between antiMSP-7 IgG2 antibodies and resistance identified in the present study is the first demonstration that antiMSP-7 responses may play a role in human resistance to P. falciparum. PfEMP-2, or mature erythrocyte surface antigen, is exported from the intraerythrocytic parasite to the erythrocyte membrane cytoskeleton, where it binds via a 19-aa sequence to band 4.1 [34, 35]. No animal or human studies have linked immune responses to PfEMP-2 with resistance to malaria.
Recently, Topolska et al. characterized a P. falciparum cDNA [25] cloned by pooled human antibody that had been obtained from malaria-exposed individuals [36] and affinity purified on parasite membrane proteins that were isolated by Triton X-114 temperature-dependent phase separation [37]. This cDNA encodes RAMA, a rhoptry protein, which is synthesized as a 170-kDa precursor during rhoptry biogenesis. In mature rhoptries, RAMA exists as a 5560-kDa protein anchored to the inner face of the rhoptry membrane via a glycosyl-phosphatidyl inositol anchor. This anchored form is discharged by free merozoites and binds to the surface of erythrocytes via the carboxy terminal region (RAMA-E; 759840 aa) and an uncharacterized RBC receptor. AntiRAMA-E IgG1 and IgG3 antibody prevalences and levels, but not those of antiRAMA-D (482758 aa), were significantly higher in uninfected than in infected individuals living in an area of Vietnam where P. falciparum is endemic [38].
Using our differential screening approach, we identified RAMA-pr (582767 aa) as the target of protective IgG1 antibodies. In our study, RP and SP did not differentially identify RAMA-E, even though PCR analysis identified this sequence in the cDNA library (data not shown). There are several possible explanations for these discrepant results, including (1) the location of the protective epitope (within the 8-aa overlap [759767 aa] between RAMA-E and RAMA-pr), (2) the lack of expression of RAMA-E in our cDNA library, (3) the dramatic differences in malaria transmission between the study sites (44% reinfection within 6 months in Vietnam vs. 50% reinfection within 5 weeks in Kenya), (4) measurement of antibody levels when all subjects were aparasitemic versus heterogeneously parasitemic, (5) host immunogenetic differences related to ethnicity, and (6) differences in recombinant proteins and antibody detection methods used in the 2 studies. Despite these differences, both studies identified that cytophilic anti-RAMA IgG antibodies are related to resistance to P. falciparum infection.
Our experiments demonstrated that RAMA-pr, a region of RAMA not involved in RBC binding, is capable of eliciting protective antibody responses. We hypothesize that antiRAMA-pr antibodies inhibit RBC invasion, and this inhibition may be mediated by several possible mechanisms, including (1) sterically interfering with RAMA-E binding to its RBC receptor, (2) blocking the interaction of RAMA-pr with a putative parasite-derived binding partner, or (3) inhibiting RAMA-RAMA association on the surface of the RBC. This cytophilic class of antibodies could also mediate parasite neutralization through an antibody-dependent cellular inhibitionbased mechanism [39]. Experiments are under way to evaluate these possible mechanisms of RAMA-prmediated resistance to infection.
In summary, we developed an approach to rationally identify new P. falciparum vaccine candidates on the basis of protective, naturally acquired human antibody responses. We identified 4 P. falciparum proteins that were uniquely recognized by antibodies in the plasma of resistant individuals but not of susceptible individuals. One of these proteins, RAMA-pr, is the target of IgG1 antibodies, which predict resistance to parasitemia. After antimalarial treatment, individuals with detectable antiRAMA-pr IgG1 antibodies had a 1.7-fold reduced risk of becoming parasitemic, 43% lower density of parasitemia, and significantly longer times to reinfection, compared with individuals without detectable antiRAMA-pr IgG1 antibodies. These data, coupled with the known role that RAMA plays in erythrocyte invasion, confirm the utility of the vaccine identification approach and underscore the potential of RAMA as a vaccine candidate for P. falciparum malaria.
Acknowledgments
This study is a secondary analysis of data that were collected in 1997 with support from the Military Infectious Disease Research Program of the US Department of Defense, the National Research Council, and the American Society of Tropical Medicine and Hygiene/Becton Dickinson Fellowship. We gratefully thank Charles Vaslet and Gretchen Langdon for research assistance. We also thank MR4 for providing us with the P. falciparum cDNA expression library contributed by D. Chakrabarti.
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